31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Characterization of novel GABAA receptor-associated proteins

The γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the mammalian brain. In order to begin to understand the mechanisms that direct the targeting and localization, clustering, and trafficking of brain GABAARs, I sought to identify cytosolic proteins that interact with the intracellular portion of the GABAAR subunits. Therefore, I used the yeast two-hybrid technique to screen an adult rat brain cDNA library using, as bait, the large intracellular loop (IL), of the GABAAR β3 subunit. I found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2), a protein known to be involved in vesicular and protein trafficking, interacts with the β subunits of the GABAA receptors. The native BIG2 and GABAARs both coprecipitated from detergent extracts with either anti-GABAAR or anti-BIG2 antibodies. Double label immunofluorescence of cultured hippocampal neurons showed that BIG2 concentrates in the trans-Golgi network and is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABAARs. The results are consistent with the hypotheses that the interaction of BIG2 with the GABAAR β subunits plays a role in the exocytosis and trafficking of assembled GABA AR to the cell surface. ^ I also isolated a clone corresponding to a novel splice form of the glutamate receptor interacting protein 1 (GRIP1). This splice form, called GRIP1c 4–7, contains 4 PDZ domains that are identical to PDZ domains 4–7 of GRIP1. GRIP1c 4–7 also contains 35 amino acids at the N-terminus and 12 amino acids at the C-terminus that differ from those of GRIP1a/b. Based on these peptide sequences, I have also isolated additional long and short GRIP1 splice variants called GRIP1d and GRIP1e 4–7. GRIP1c 4–7 interacted with GluR2/3 subunits of the AMPA receptor and with gephyrin but not with the GABA AR α1 subunit in detergent extracts of rat brain membranes. In low-density hippocampal cultures, GRIP1c 4–7 clusters colocalized with components of both GABAergic and glutamatergic synapses. GRIP1c 4–7-specific antibodies recognized a 75 kDa Mr protein that is enriched in a postsynaptic density (PSD) fraction isolated from brain. These results indicate that GRIP1c 4–7 plays functional role(s) in both GABAergic and glutamatergic synapses.

Biallelic mutations in PDE10A lead to loss of striatal PDE10A and a hyperkinetic movement disorder with onset in infancy

Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species